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研究生:陳嘉榮
研究生(外文):Chia-Jung Chen
論文名稱:乳牛結核病檢驗方法之評估
論文名稱(外文):Evaluation of the Diagnostic Methods for Bovine Tuberculosis in Dairy Herds
指導教授:吳永惠吳永惠引用關係
指導教授(外文):Yeong-Huey Wu
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:獸醫學系所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:75
中文關鍵詞:牛結核病牛分支桿菌牛結核病之診斷方法
外文關鍵詞:Bovine tuberculosisMycobacterium bovisDiagnostic methods for bovine tuberculosis
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為評估乳牛各種牛結核病檢驗方法之實用性,對皮內結核菌素試驗 (intradermal tuberculin test, ITT) 陽性場16場87頭及陰性場8場40頭乳牛,於ITT檢驗後2週內,採血以市面新開發之Mycobacterium tuberculosis complex核酸探針冷光儀偵測法 (reliable assay protocol for identification of diseases - bioactive amplification with probing for Mycobacterium tuberculosis complex, RAPID BAP-MTB) 和Mycobacterium bovis抗體酵素免疫結合吸附法 (enzyme - linked immunosorbent assay for Mycobacterium bovis antibody, Bovine TB Ab-ELISA),以及傳統之迦瑪干擾素試驗 (gamma interferon assay, INF-γ assay) 和巢式聚合酶連鎖反應 (Nested-polymerase chain reaction, Nested - PCR) 進行檢驗,採鼻黏液行Nested-PCR及RAPID BAP-MTB,並對ITT陽性牛採咽背和/或縱膈淋巴結行M. bovis分離與鑑定,然後評估各方法之敏感性、特異性、Kappa值和實用性。結果,若以M. bovis分離鑑定結果為判定基準,則由Kappa值顯示血液樣本Nested-PCR之敏感性 (70.6 %) 和特異性 (77.8 %),比其他檢驗法 (Bovine TB Ab-ELISA、INF-γ assay及RAPID BAP-MTB),呈現最為優異;若以各檢驗法為判定基準,則顯示ITT之敏感性 (54.0-100 %) 優於其他各法,但其特異性 (31.4-47.1 %) 則除優於Bovine TB Ab-ELISA 法外,普遍相對地劣於其他檢驗法 ; 又以各檢驗法為判定基準,則ITT併用Bovine TB Ab-ELISA或INF-γ assay,比單用ITT可普遍地且明顯地提高敏感性,但其特異性則相對地下降。
In order to evaluate the practicality of diagnostic methods for bovine tuberculosis in dairy herds, the new commercial methods (reliable assay protocol for identification of diseases-bioactive amplification with probing for Mycobacterium tuberculosis complex, RAPID BAP-MTB; enzyme-linked immunosorbent assay for Mycobacterium bovis antibody, Bovine TB Ab-ELISA) and traditional methods (intradermal tuberculin test, ITT ; nested-polymerase chain reaction, Nested-PCR ; gamma interferon assay, INF-γ assay) were simultaneously conducted in 87 cows of 16 tuberculosis-infected herds and 40 cows of 8 tuberculosis-free herds within two weeks after intradermal tuberculin test (ITT). The Nested-PCR and RAPID BAP-MTB were also conducted for nasal mucus samples, and isolation of the retropharyngeal and/or mediastinal lymph nodes for M. bovis were also performed for the ITT positive cattles. Then, the sensitivities, specificities and Kappa values were evaluated. If using the results of M. bovis isolation as the diagnostic standard, the blood Nested-PCR showed the most excellent sensitivity (70.6 %) and specificity (77.8 %) than the other methods (Bovine TB Ab-ELISA, INF-γ assay and RAPID BAP-MTB) according to the Kappa values ; If using the results of each method as the diagnostic standard, ITT exhibited the most excellent sensitivities (54.0-100 %) than the other methods, but its specificities were universally lower (31.4-47.1 %) than other methods, besides the Bovine TB Ab-ELISA ; If using the results of each method as the diagnostic standard, the sensitivities of ITT combining with Bovine TB Ab-ELISA or INF-γ assay were universally and remarkably improved than ITT used alone, but their specificities were relatively decreased.
中文摘要....................................................I
Abstract.................................................III
謝誌.......................................................V
目錄......................................................VI
圖表目錄....................................................X
第1章 前言..................................................1
第2章 文獻回顧..............................................3
2.1 牛結核病發生概況....................................3
2.2 牛結核病之傳染途徑..................................4
2.3 牛結核病之致病機轉..................................5
2.4 牛結核病之免疫反應..................................5
2.5 牛結核病難以清除之原因..............................7
2.6 牛結核病之動物生前診斷方法...........................8
2.6.1 皮內結核菌素試驗 (intradermal tuberculin test,
ITT)...........................................9
2.6.2 迦瑪干擾素檢驗 (gamma interferon assay, IFN-γ
assay)........................................11
2.6.3 聚合酶連鎖反應檢驗 (polymerase chain reaction,
PCR)..........................................12
2.6.4 M. tuberculosis complex核酸探針冷光儀偵測法
(reliable assay protocol for identification
of diseases-bioactive amplification with
probing for M. tuberculosis complex, RAPID BAP-
MTB)..........................................13
2.6.5 M. bovis抗體酵素免疫結合吸附法 (enzyme-linked
immunosorbent assay for M. bovis antibody,
Bovine TB
Ab-ELISA).....................................14
第3章 材料與方法...........................................15
3.1 試驗設計及供試牛隻.................................15
3.2 皮內結核菌素檢驗 (intradermal tuberculin test,
ITT).............................................15
3.2.1 診斷液........................................15
3.2.2 檢驗及判定.....................................15
3.3 迦瑪干擾素試驗 (gamma interferon assay, IFN-γ
assay)...........................................16
3.3.1 試劑..........................................16
3.3.2 試劑使用前置處理................................16
3.3.3 檢驗..........................................17
3.3.4 判讀..........................................18
3.4 巢式聚合酶連鎖反應 (nested-polymerase chain reaction,
Nested-PCR)......................................18
3.4.1 核酸萃取之試劑及儀器............................18
3.4.2 標準菌株之核酸萃取..............................19
3.4.3 血液樣本之採集及核酸萃取.........................19
3.4.4 鼻黏液樣本之採集及核酸萃取.......................20
3.4.5 聚合酶連鎖反應引子對之設計.......................21
3.4.6 聚合酶連鎖反應之試劑及儀器.......................21
3.4.7 聚合酶連鎖反應之核酸增幅.........................22
3.4.8 巢式聚合酶連鎖反應之核酸增幅.....................22
3.4.9 增幅產物之分析.................................23
3.4.10 增幅產物之純化與定序............................23
3.5 M. bovis抗體酵素免疫結合吸附法 (enzyme-linked
immunosorbent assay for M. bovis antibody, Bovine
TB Ab-ELISA).....................................23
3.5.1 套組檢驗方法...................................24
3.5.2 判讀..........................................24
3.6 M. tuberculosis complex核酸探針冷光儀偵測法
(reliable assay protocol for identification of
diseases-bioactive amplification with probing for
M. tuberculosis complex,RAPID BAP-MTB)...........24
3.6.1 臨床樣本之DNA萃取..............................24
3.6.2 核酸增幅.......................................25
3.6.3 雜合反應.......................................25
3.6.4 判讀..........................................26
3.6.5 判讀標準.......................................26
3.7 結核桿菌分離與鑑定.................................26
第4章 結果.................................................28
4.1 PCR及Nested-PCR之敏感性分析.......................28
4.2 PCR及Nested-PCR之特異性分析.......................29
4.3 以結核菌分離為診斷標準分析各檢驗法之實用性............31
4.3.1 M. bovis抗體酵素免疫結合吸附法
(Bovine TB Ab-ELISA)..........................31
4.3.2 IFN-γ試驗.....................................31
4.3.3 血液樣本Nested-PCR.............................32
4.3.4 血液樣本RAPID BAP-MTB..........................33
4.3.5 鼻黏液樣本Nested-PCR...........................33
4.3.6 鼻黏液樣本RAPID BAP-MTB........................34
4.3.7 由敏感性、特異性、Kappa值和操作簡便性比較各檢驗
法之實用性.....................................35
4.3.8 單以分離鑑定為診斷標準之缺失.....................35
4.4 以各種生前結核病檢驗法互為診斷基準之比較..............36
4.4.1 ITT...........................................36
4.4.2 Bovine TB Ab-ELISA............................36
4.4.3 IFN-γ 試驗....................................36
4.4.4 血液Nested-PCR................................36
4.4.5 鼻黏液Nested-PCR..............................38
4.4.6 血液RAPID BAP-MTB.............................38
4.4.7 鼻黏液RAPID BAP-MTB...........................38
4.4.8 綜合判定.......................................38
4.5 各檢驗法與ITT併用時之實用性評估.....................39
4.5.1 併用Bovine TB Ab-ELISA時......................39
4.5.2 併用IFN-γ時...................................40
4.5.3 併用血液Nested-PCR時...........................40
4.5.4 併用鼻黏液Nested-PCR時.........................42
4.5.5 併用血液RAPID BAP-MTB時........................42
4.5.6 併用鼻黏液RAPID BAP-MTB時......................42
4.5.7 綜合判定.......................................43
4.6 ITT腫脹程度之分析.................................43
4.7 IFN-γ試驗之OD (M. bovis) 值與分離培養之關係.........43
4.8 Nested-PCR產物定序結果............................44
第5章 討論.................................................47
參考文獻...................................................52
附錄......................................................64
縮寫表....................................................74
作者簡介...................................................75
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